Estudo das interações da miltefosina com membranas de L. (Leishmania) amazonensis e macrófagos peritoneais
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2016-02-15
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Universidade Federal de Goiás
Resumo
Miltefosine (MT) is a alkylphospholipid originally developed for treatment
of breast cancer and other solid tumors. It is currently used in the treatment of
leishmaniasis, an infectious parasitic disease caused by protozoa of the genus
Leishmania, being the first oral drug adopted for this purpose. However, its
mechanism of action remains unclear. Electron paramagnetic resonance (EPR)
spectroscopy of a spin-labeled lipid (5-DOXIL stearate) and a thiol-specific spin
label (4-maleimido-TEMPO) in the membrane of axenic amastigotes of
L.(Leishmania) amazonensis and peritoneal macrophages from Balb/c mice
showed that MT causes significant increase in membrane dynamics at similar
concentrations that inhibit parasite growth or are cytotoxic to macrophage.
Although these alterations can be detected using a spin-labeled lipid, our
experimental results indicated that MT interacts predominantly with the protein
component of the membrane. Using a method for the rapid incorporation of MT
into the membrane, these effects were measured immediately after treatment.
Cytotoxicity, estimated via microscopic counting of living and dead cells,
indicated ~80% parasites and macrophages death at the concentration of MT at
which EPR spectroscopy detected a significant change in membrane dynamics.
Cell viability, analyzed using the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-
diphenyltetrazolium bromide tetrazolium) reduction assay, showed that 50%
inhibitory concentration (IC50) of MT depends on the cell concentration used in
the assay. This dependence was analyzed using a theoretical equation
involving biophysical parameters such as the partition coefficient of watermembrane
and MT concentrations on the membrane and in the aqueous
medium. The data showed that cells more sensitive to MT are respectively:
erythrocytes, Leishmania promastigotes and Leishmania amastigotes and
macrophage. The IC50 value of MT for 4 x 107 parasites/mL was 24,35 M. For
the same cell concentration, a significant alteration was detected in the
membrane lipid fluidity of parasites to 15 M of MT. The EPR spectra of spinlabeled
membrane-bound proteins were consistent with more expanded and
solvent exposed protein conformations, suggesting a detergent-like action, with
a possible formation of micelle-like structures around polypeptide chains.
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FERNANDES, K. S. Estudo das interações da miltefosina com membranas de L. (Leishmania) amazonensis e macrófagos peritoneais. 2016. 146 f. Tese (Doutorado em Física) - Universidade Federal de Goiás, Goiânia, 2016.